Gene expression of the alpha subunit of a human translation initiation factor eIF-2, has been shown to be regulated during the mitogenic activation of quiescent T cells. Previous work has revealed that eIF-2alpha is a single copy housekeeping gene with multiple start sites. Its promoter region is GC rich with five hypersensitive sites identified. Within one of these hypersensitive sites, a palindromic element was identified, which binds a transcription factor, alpha-Pal. Mutations of this element have been shown to affect eIF-2alpha promoter activity in a luciferase reporter gene construct transduced into NIH 3T3, 293 and COS cells. Downstream of the transcription start sites, an Inr (initiator) sequence has been identified that was oriented to allow transcription in the opposite direction to the sense transcript. The presence of this "antisense" transcript has been demonstrated by RT-PCR in Go T cells, the level of which becomes reduced upon mitogenic stimulation of the cells. We have continued the analysis of the promoter region of eIF-2alpha. Region -560 to +1147 was cloned into a luciferase reporter gene construct in either the sense or antisense (opposite) orientation and transfected into NIH 3T3, 293, COS or Jurkat cells. Analysis of luciferase activity showed weak activity for the sense constructs and strong activity for the antisense constructs. These results support a major role of antisense transcription in the regulation of eIF-2 gene expression. Deletion mutational analysis from either the 5' or 3' ends of either construct revealed that besides the alpha-Pal sites and Inr regions, there are other potential regulatory cis acting regions. We have evaluated the significance of overlapping alpha-Pal and E-box binding sites. In the tandem repeat alpha-Pal binding motif noted, it appears likely that alpha-Pal and MAX heterodimers may modulate transcriptional activation in the sense orientation. An AP1 site has been identified by footprint analysis which is located at +91, the site of the first exon/intron junction, within the first intron of the eIF-2alpha gene. This AP1 region showed increased gel shift activity with nuclear extracts isolated from activated T cells that can be supershifted with cfos and cJun/JunD antibodies. Reporter gene constructs containing this region can be induced with PMA in HL-60 cells. Mutations of critical G residues reduces this induction. We have also identified a new downstream region (Inr) that binds nuclear factors. Gel-shift analysis with a variety of nuclear extracts from a variety of cells reveals binding activity which can be competed specifically with cold probe, but not with mutant probe. This region, however, when mutated reduced the antisense activity by only 20%. Deletion of the region surrounding the IRS sequence in the sense constructs increased its activity by 2.5 fold. PCR primers based upon the human eIF-2alpha sequence were generated and used to produce an equivalent PCR product from mouse genomic DNA. This product was cloned and sequenced and found to be 74 percent homologous to the human sequence. By PCR screening of a mouse BAC library, a mouse genomic clone has been obtained and will be used in sequence comparison and functional studies.